Prevalence of Homologous Recombination Deficiency Among Patients With Germline RAD51C/D Breast or Ovarian Cancer

Key Points Question What is the prevalence of homologous recombination deficiency (HRD) in tumors from patients with germline RAD51C/D breast and ovarian cancer? Findings In this cohort study, the prevalence of HRD based on genomic and functional tumor biomarkers in germline RAD51C/D carriers was less than 70%. All estrogen receptor–positive breast cancers lacked HRD, in part associated with the retention of the wild-type allele in RAD51C/D. Meaning These findings highlight the importance of HRD testing to guide therapeutic decision-making for patients with RAD51C/D-associated cancer.


Introduction
RAD51C and RAD51D are RAD51 paralogs involved in the homologous recombination repair (HRR) of double-stranded DNA breaks.Together with other RAD51 family members, they form protein complexes (BCDX2 and CX3) that act within the BRCA1/2-dependent HRR pathway and contribute to genomic stability.Germline pathogenic variants (PVs) in RAD51C (OMIM 602774) and RAD51D (OMIM 602954) (RAD51C/D) are expected to cause homologous recombination deficiency (HRD) and genomic instability when there is biallelic inactivation, mainly through gene-specific loss of heterozygosity (gsLOH).[11] Current methods to assess HRD fall into 3 categories: HRR gene panel sequencing, genomic scars and signatures, and functional assays. 12Selection of patients for treatment with a poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitor is currently based on germline BRCA1/2 (BRCA1, OMIM 113705; BRCA2, OMIM 600185) mutation status for breast cancer or platinum sensitivity, BRCA1/2 mutation, or genomic HRD for ovarian cancer. 12[15][16][17][18][19][20] The prevalence of genomic HRD in tumors of RAD51C/D PV carriers has mainly been investigated within large cohorts of pan-cancer HRD analysis. 21In a small sample, Li et al 22 showed that 7 of 9 cases of RAD51C-associated breast cancer (77.8%) harbored genomic HRD based on a high genomic instability score (GIS) and concomitant gsLOH.In ARIEL2, Swisher et al 23,24 showed that mutations in RAD51C/D were associated with genomic HRD (based on high genomic LOH) and response to the PARP inhibitor rucaparib in 5 of 7 patients (71.4%) with relapsed high-grade ovarian cancer, reaching a median progression-free survival similar to patients with mutated BRCA1/2.
Similarly, one study showed a high sensitivity to DNA-damaging chemotherapy in a patient with breast cancer with a RAD51D germline PV and functional HRD. 25 Overall, prior clinical trials in breast cancer or ovarian cancer have analyzed the efficacy of platinums and PARP inhibitors for patients with germline RAD51C/D PVs observing a wide range of treatment responses. 26,27Some studies have reported the presence of gsLOH 26 but lack of concordance with HRD by GIS, and others do not report biallelic inactivation or HRD status. 28In summary, prior evidence highlights the necessity of knowing the HRD functional status of RAD51C/D germline carriers with cancer to determine whether they might benefit from targeted therapeutic management.We aimed to perform a comprehensive molecular analysis of a large cohort of patients with RAD51C/D untreated primary breast cancer and ovarian cancer to describe the prevalence of HRD by different biomarkers and investigate the role of the germline alterations in tumorigenesis.

Study Population
Between January 1, 2014, and December 31, 2021, 9507 individuals from 18 hereditary cancer units across Spain underwent germline genetic testing for breast cancer and/or ovarian cancer

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Homologous Recombination Deficiency Among Patients With RAD51C/D Breast or Ovarian Cancer predisposition.This retrospective cohort study included women and men with RAD51C or RAD51D germline PVs, as well as family members carrying these variants.We did not check for sample size using a power analysis because our study included all patients older than 18 years tested routinely in screening programs.All participants provided written informed consent before study entry and were codified by their respective center.Variants were classified by each independent laboratory and subsequently reviewed by the central laboratory according to the American College of Medical Genetics and Genomics guidelines. 29e carrier frequency for RAD51C/D PVs was calculated as the number of index patients with a PV in RAD51C/D divided by the total number of index patients tested for RAD51C/D.

HRR Assays
Formalin-fixed, paraffin-embedded (FFPE) tumor samples were requested from the participating centers in 2022, and HRD analyses were performed from June 2022 to February 2023.

RAD51 Immunofluorescence Test
To evaluate the functional HRR status with the RAD51 test, FFPE whole tumor sections (3 μm) from early untreated breast cancer and ovarian cancer were used to detect RAD51 foci (as a functional readout of HRD), γH2AX foci (as a biomarker of double strand DNA breaks), and BRCA1.Each biomarker was counterstained with geminin (as a marker of S/G2 cell cycle phase) and DAPI (4′,6-diamidino-2-phenylindole).Commercially available primary and secondary antibodies were used as per the protocol in a previous study. 17The scoring was performed blindly onto life images using a ×60-immersion oil objective in a Nikon Ti2-Eclipse microscope.At least 40 geminin-positive cells were analyzed per sample, and γH2AX scoring was used as a quality check to ensure the presence of endogenous DNA damage to evaluate HRR functionality (cutoff: 25% geminin-positive cells with γH2AX foci).6][17] Functional HRD was defined by low RAD51 scores (Յ10%), and functional homologous recombination proficiency (HRP) by high RAD51 scores (>10%).

Gene Sequencing and Genomic Instability
1][32] Tumor DNA was isolated from FFPE samples and used for targeted multiplex polymerase chain reaction amplification and library construction.Next-generation sequencing (Illumina) was conducted to screen tumor mutations of BRCA1 and BRCA2 and 13 additional genes relevant to DNA repair (ATM [OMIM 607585], BARD1 A standardized bioinformatic analysis was used to determine the GIS based on loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions. 33Genomic HRD was defined as a GIS of 42 or higher.To estimate the gsLOH status of the RAD51C/D loci and other HRR genes, the computationally most likely allele-specific copy number at each single-nucleotide variation location was analyzed.

Statistical Analysis
A descriptive analysis was performed to describe the study population.

Clinical Characteristics of RAD51C/D-Associated Breast and Ovarian Cancers
The clinical characteristics of patients with RAD51C/D breast cancer are summarized in
Moreover, 62.5% of tumors (5 of 8) with low GIS retained the wild-type allele (non-gsLOH), which could explain the lack of an HRD profile (Figure 1C and A).
The concordance between genomic and functional HRD was 91% (Cohen κ = 0.8 [95% CI, 0.5-1]; P < .001)(Figure 1D; eFigure 4 in Supplement 1), with 63.6% of tumors (14 of 22) harboring HRD by both techniques and 27.3% (6 of 22) showing HRP.The concordance between gsLOH and GIS was 76%, and between gsLOH and RAD51, it was 83% (eFigure 4 in Supplement 1).Tumors with non-gsLOH in RAD51C showed HRP, with RAD51 foci formation and low GIS.Discordancy was observed in 1 ovarian cancer case with a germline RAD51D PV, which showed borderline results for both genomic instability and RAD51 foci (GIS of 42 and 13% RAD51).The other case was a surgical ovarian cancer specimen with a germline RAD51C PV, showing HRD by GIS (81) and HRP by RAD51 (32%).Overall, functional and genomic HRD were highly concordant and ranged between 55% and 90% depending on the gene and type of tumor.

Association of HRD With Age and Cancer Subtype
We investigated whether lack of HRD was more common in patients with an older age (>50 years) at onset, suggesting that their tumors were of sporadic vs hereditary origin.However, we found no significant association between age at diagnosis and HRD by RAD51 or gsLOH (eFigure 5A-C in Supplement 1).Finally, we stratified the results by cancer subtypes, namely ER-positive breast cancer, ER-negative breast cancer, and high-grade ovarian cancer, as all ovarian cancer samples analyzed were of high grade (Figure 2; eFigure 5D-E in Supplement 1).One of the RAD51 high ER-negative breast cancer cases was an ERBB2-positive tumor (Figure 1A).Estrogen receptor-positive breast cancer had a higher prevalence of HRP and concomitant non-gsLOH compared with ER-negative breast cancer and high-grade ovarian cancer (Figure 2B).

Discussion
To In this study of 9507 index patients, the prevalence of an RAD51C/D PV was 1.0%, slightly higher than in population-based studies. 1,2Almost half of the index patients had no family history of breast cancer or ovarian cancer, compatible with the moderate cancer risk associated with these gene alterations. 3One variant (RAD51D c.694C>T) was highly prevalent in our cohort (57.1%), and although it had previously been reported elsewhere, 34  Regarding age, we did not find any correlation between an earlier age at onset and a higher occurrence of HRD.The majority of ovarian cancers showed HRD associated with gsLOH, as previously reported 35,36 and like ER-negative breast cancer.We found that all ER-positive breast cancer cases were HRP by RAD51 foci and lacked gsLOH.This finding is consistent with pan-cancer studies reporting moderate rates of biallelic inactivation among RAD51C/D cases compared with high rates in BRCA1/2. 37This finding also suggests that ER-positive breast cancer in patients with germline PVs in RAD51C/D might, in fact, be sporadic tumors.Similarly, Li et al 22 found that 2 ER-positive cases out of 9 cases of breast cancer retained heterozygosity across the RAD51C locus and were the only cases of breast cancer that did not exhibit HRD.Our findings suggest that germline RAD51C/D PV is not associated with the tumorigenesis of ER-positive breast cancer, consistent with epidemiologic data showing that germline RAD51C/D PV carriers have a higher risk of developing ER-negative breast cancer.

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Homologous Recombination Deficiency Among Patients With RAD51C/D Breast or Ovarian Cancer of clinical and molecular features for the 45 breast cancer (BC) or ovarian cancer (OC) tumors analyzed.Waterfall with the RAD51 scores (bars) and yH2AX scores (dots) for each sample.The next studied whether the functional HRD status of RAD51C/D tumors varied across PVs.Different tumors with the same PV showed variable HRD status, regardless of cancer type (eFigure 3 in Supplement 1).In particular, functional HRD values varied in tumors with the following PVs in RAD51C: deletion of exons 4 to 9, c.705+1G>A, c.709C>T, c.965+5G>A, c.979_989dup, and c.1026+5_1026+7del; and in RAD51D, c.94_95del and c.694C>T.

Figure 2 .
Figure 2. Distribution of Functional Homologous Recombination Deficiency (HRD) Across Tumor Subtypes 100 This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline and was reviewed and approved by, and conducted according to, the ethical standards of the Vall d'Hebron Hospital Ethics Committee and all institutional review boards of the participating centers (Catalan Institute of Oncology, Hospital Universitari Parc Taulí, Hospital Miguel Servet de Zaragoza, Clinical University Hospital Virgen Arrixaca, Arnau de Vilanova University Hospital, Hospital San Pedro de Alcántara, Clínico Universitario de Valencia, Hospital General Universitario de Ciudad Real, Xerencia de Xestión Integrada de A Coruña, Hospital Universitario de Galdakao, Hospital de la Santa Creu i Sant Pau,
Continuous variables were expressed as median (IQR) values, and categorical variables were expressed as absolute values and percentages.The Cohen κ coefficient was used to analyze the concordance between HRD assays.The association among HRD, gsLOH, specific tumor subtype, and age at diagnosis was evaluated using the t test, univariate logistic regression, or univariate logistic regression with the Firth bias reduction method (to solve the problem of perfect separation).All P values were from 1-sided tests and results were deemed statistically significant at P < .05,and95%CIswerereported.Analyses were performed with R statistical software, version 4.1.1(RProjectforStatisticalComputing).Genetic susceptibility to breast and/or ovarian cancer was assessed for 9507 index patients.Among them, 91 had a PV in RAD51C/D.Furthermore, the study encompassed 90 family members with a germline PV in RAD51C/D.In total, 181 individuals were included, with 113 carrying RAD51C PVs and 68 carrying RAD51D PVs (Table1).A total of 157 carriers (86.7%) were women and 181 (55.8%) had received a diagnosis of cancer, primarily breast cancer or ovarian cancer.Additional details of the study population are presented in Table1.

Table 2 .
Characteristics of RAD51C/D-Associated Breast CancersHomologous Recombination Deficiency Among Patients With RAD51C/D Breast or Ovarian Cancer tumors, and 41.7% (15 of 35) had triple-negative breast cancer.Among 68 RAD51D carriers, 20.6% (14 of 68) had received a diagnosis of breast cancer, and 1 woman had a second primary breast cancer.The median age at diagnosis was 38 years (IQR, 35-41 years).All tumors but 1 were invasive ductal carcinoma, and 66.7% (10 of 15) were diagnosed at stages I or II.The distribution of hormonal receptor status was also similar between the 2 genes, with 53.3% (8 of 15) of RAD51D breast cancers being ER negative and 46.7% (7 of 15) being triple-negative breast cancers.
14breviations: ER, estrogen receptor; NA, not available; PR, progesterone receptior.aOnlyfirstbreastcancerdiagnosis.JAMA Network Open | OncologyAssessment of the HRD StatusOf 181 patients, 98 had breast cancer and/or ovarian cancer.From those, we obtained 45 untreated FFPE tumor samples (23 breast cancer and 22 ovarian cancer) to evaluate the HRD status (eFigure 1 in Supplement 1).Two samples with insufficient tumor content and 15 samples with insufficient tissue material or DNA were excluded from the functional and genetic or genomic HRD analyses, respectively.The RAD51 foci test was successful in 88.4% of samples (38 of 43).Five samples were nonevaluable due to poor tissue quality.The Myriad myChoice HRD test was successful in 93.3% of samples (28 of 30).Two samples were nonevaluable for GIS due to poor DNA quality, although they were evaluable for HRR gene mutation calling and gsLOH status (eFigure 1 in Supplement 1).All germline PVs in RAD51C and RAD51D were identified in the respective tumors.Panel sequencing of HRR-related genes additionally identified 1 tumor with a likely BRCA1 PV with gsLOH, 2 with BRCA2 PVs with gsLOH, and 1 tumor with a PV in PALB2 without gsLOH (Figures 1A).All germline RAD51C/D tumors had high levels of nuclear BRCA1 foci, which excluded potential concomitant epigenetic silencing of BRCA1 as the origin of HRD,14except for 1 RAD51C carrier with low levels of BRCA1 foci

Table 3 .
Characteristics of RAD51C/D-Associated Ovarian Cancers JAMA Network Open.2024;7(4):e247811.doi:10.1001/jamanetworkopen.2024.7811(Reprinted) April 22, 2024 6/14 Downloaded from jamanetwork.comby guest on 04/24/2024 table indicates the type of each tumor, gene mutated, gene-specific loss of heterozygosity (gsLOH) status, genomic instability score (GIS), and age at diagnosis.B, Functional HRD by RAD51 in hereditary cancers.The RAD51 scores of 141 tumor samples from patients with BC or OC with germline pathogenic variants in RAD51C, RAD51D, BRCA1, BRCA2, or PALB2 are shown.C, Genomic HRD by genomic instability.The GIS of 28 tumor samples from patients with BC or OC with germline pathogenic variants in RAD51C or RAD51D are shown.The gsLOH status in RAD51C/D is also shown.D, Correlation between RAD51 and GIS, showing a 91% concordance.Each dot represents 1 tumor per patient, gray lines indicate the mean of each group, and the horizontal dotted lines indicate the predefined threshold of the RAD51 test (10%) or GIS (42) to discriminate HRD vs homologous recombination proficiency (HRP) status.Dotted areas in panel D represent concordant HRD or HRP status by both tests.Het indicates heterozygous; HRR, homologous recombination repair; NA, not available; NE, not evaluable; and TNBC, triple-negative breast cancer.likelydue to a concomitant tumor BRCA1 PV (eFigure 2 in Supplement 1).In summary, 13.3% of tumors (4 of 30) from patients with germline RAD51C/D PVs concomitantly carried mutations in other HRR genes, and none showed epigenetic silencing of BRCA1.
our knowledge, it is currently unclear whether patients with germline PVs in RAD51C/D can benefit from DNA damage repair-targeted agents, such as PARP inhibitors.Homologous recombination deficiency, mainly occurring in mutated BRCA1/2 tumors, has been shown to be a potent biomarkerJAMA Network Open | Oncology its high frequency may suggest a founder origin.Within this cohort, we further characterized 113 individuals who carried a germline RAD51C PV and 68 individuals who carried a germline RAD51D PV.Half the individuals had received a diagnosis of cancer, primarily breast or ovarian cancer.The clinical characteristics of breast cancer or ovarian cancer were similar between carriers of RAD51C and carriers of RAD51D.Breast cancer cases were enriched for ER-negative phenotype (52.8%), an aggressive tumor type lacking targeted therapies apart from the use of PARP inhibitors for patients with germline BRCA1/2 PVs.A total of 19.6% of patients with ovarian cancer received a diagnosis before 50 years of age, the majority at an advanced disease stage, which highlights the importance of preventive oophorectomy for female carriers of RAD51C/D.The incidence of HRD in germline RAD51C/D was lower than in germline BRCA1/2 or PALB2, especially among breast cancer samples.We investigated the potential explanation for the lower HRD frequency, including the type of mutation, age at diagnosis, gsLOH, or ER status.Different tumors with the same PV displayed contrasting HRD statuses, indicating no correlation between the PV type and HRD.